Sequence of events leading to membrane fusion during acrosomal exocytosis in human spermatozoa

DE BLAS, G; ROGGERO, C; BELMONTE, S; TOMES, C; MAYORGA, LS.

Pucón, Chile

Simposio; 13th INTERNATIONAL SYMPOSIUM OF CHROMAFFIN CELL BIOLOGY; 2006

The acrosome is a membrane-limited granule which overlies the nucleus of the mature spermatozoon. In response to stimuli, it undergoes a special type of calcium-dependent exocytosis termed the acrosome reaction which is an absolute prerequisite for fertilization.  SNARE complexes play a central role in most membrane fusion events.  Acrosomal exocytosis has several unique features that allow for a detailed characterization of the dynamics of assembly and disassembly of SNARE complexes and its regulation during membrane fusion.  It is an all-or-nothing event that occurs as a synchronized wave, with no recycling of components and requires neurotoxin-sensitive SNAREs.  By using a functional assay and immunofluorescence techniques in combination with neurotoxins and a photosentitive Ca2+ chelator, we have shown that in resting sperm, SNAREs are associated in cis complexes.  Upon Ca2+ entrance into the cytoplasm, Rab3A is activated and anchored to the membranes in a process that is modulated by cholesterol.  Interestingly, this sterol does not affect late steps of the membrane fusion process1.  Active Rab3A triggers the disassembly of cis SNARE complexes by means of NSF and aSNAP.  SNAREs are now free to re-assemble in loose trans complexes that are resistant to NSF/aSNAP action.  At this point, Ca2+ must be released from inside the acrosome to initiate the final steps of membrane fusion that require fully assembled trans SNARE complexes and synaptotagmin VI2. The function of this Ca2+-sensoring protein is regulated by PKC-mediated phosphorylation of a conserved polybasic domain necessary for interaction with multiple effectors.  Synaptotagmin desphosphorylation is probably an early event during exocytosis that allows the interaction of this protein with other components of the fusion machinery3.  Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drives membrane fusion during the acrosomal exocytosis of human spermatozoa. 1.       Belmonte SA et al. (2005) Develop. Biol. 285, 393-408. 2.       De Blas GA et al. (2005) PLoS Biology. 3, e323 3.       Roggero CM et al. (2005) Develop. Biol. 285, 422-435. Supported by Howard Hughes Medical Institute, CONICET (Argentina) and ANPCyT (Argentina)